Selective κ opioid antagonists nor-BNI, GNTI and JDTic have low affinities for non-opioid receptors and transporters.

BACKGROUND: Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets. RESULTS: In binding assays, the three antagonists showed no detectable affinity (K(i)≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over µ and δ (3 to 44-fold). Nor-BNI bound weakly to the α(2C)-adrenoceptor (K(i) = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α(1A)-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (K(B) = 3.7 µM). JDTic bound to the noradrenaline transporter (K(i) = 54 nM), but only weakly inhibited transport (IC50 = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K(i) = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers. CONCLUSIONS: Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α(1A)-adrenoceptors). This may contribute to GNTI's severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor.

INTRODUCTION: P-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood-brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1-40) HiLyte Fluor™ TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine. METHODS: Male Wistar rats' everted intestines were divided into sacs, each sac was filled with 10µM Ab-HiLyte with or without 50µM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594nm at each appropriate time. RESULTS: The Ab-HiLyte P-gp mediated efflux had a K=1.00×10(-2)min(-1) and t(1/2)=68.74min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K=1.65×10(-3)min(-1)) and the half life is extremely increased (t(1/2)=419min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K=1.81×10(-2)min(-1) and t(1/2)=38.28min). DISCUSSION: The results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.

Modulation and absorption of xenobiotics: the synergistic role of CYP450 and P-gp activities in cancer and neurodegenerative disorders.

P-glycoprotein (P-gp) is involved in MDR and in neurodegenerative disorders such as Parkinson disease (PD), Alzheimer disease (AD) and epilepsy. Cytochrome P450 enzymes (CYP450s) catalyze the metabolism of a wide variety of endogenous and exogenous compounds including xenobiotics, drugs, environmental toxins, steroids, and fatty acids. P-gp substrates, inhibitors and inducers should be designed and developed studying interacting mechanism with both P-gp an CYP450 enzymes before they could be employed in MDR and/or in neurodegenerative disorders. Here, the ex vivo rat everted gut sac assay has been proposed as an immediate approach to simultaneously study metabolism and transport of drugs. Elacridar, verapamil and cyclosporine A (CsA), P-gp inhibitor, substrate and modulator respectively, have been tested to validate this ex vivo approach. The new model have been used yet to develop our ligands MC18, MC266 and MC80, both as potential drugs for MDR and radiotracers for diagnosis of neurodegenerative disorders. Herein, a comparative evaluation of transport and metabolic results, by using in vitro, ex vivo and in vivo assays, is reported.

Substrates, inhibitors and activators of P-glycoprotein: candidates for radiolabeling and imaging perspectives.

In recent years, several PET tracers for monitoring the activity and expression of P-gp at the BBB have been tested. P-gp substrates such as [(11)C]verapamil and [(11)C]loperamide can be employed to visualize P-gp activity, but they display a moderate baseline uptake in the brain and formation of radiolabeled metabolites which hamper the interpretation of PET data. P-gp inhibitors such as [(11)C]elacridar, [(11)C]laniquidar and [(11)C]tariquidar have been tested to investigate P-gp expression and the results need further investigation. Recently, we developed MC18, MC266 and MC80, that have been characterized as an inhibitor, substrate and inducer of P-gp both by in vitro assays and in the everted gut sac method. These compounds have been radiolabelled with (11)C and been evaluated in vivo. In the present review, we compare the outcome of biological in vitro assays and the corresponding in vivo PET data for the P-gp inhibitors [(11)C]MC18 and [(11)C]elacridar, the P-gp substrates [(11)C]MC266 and [(11)C]verapamil, the P-gp inducer [(11)C]MC80 and the P-gp modulator cyclosporin A. Since a satisfactory overlap was found comparing in vivo results and the corresponding in vitro findings, the proposed biological in vitro assays could be predictive for the in vivo PET data of novel radiotracers. PET tracers could be employed for various purposes: radiolabeled P-gp inhibitors to monitor decreased expression of P-gp at the BBB in neurodegenerative disorders such as Alzheimer's and Parkinson's disease; and radiolabeled P-gp substrates with a high baseline uptake to monitor increased expression of P-gp in epileptic foci.

In vitro and ex vivo characterization of sigma-1 and sigma-2 receptors: agonists and antagonists in biological assays.

Methods for addressing sigma receptor affinity and activity have been explored and although several protocols have been employed, only few procedures resulted reliable. Sigma-1 receptor affinity protocol using guinea-pig brain and (+)-[(3)H]-pentazocine and sigma-2 receptor affinity protocol employing rat liver and [(3)H]-DTG are usually reported by authors as standard procedures. By contrast, the intrinsic activity evaluation of sigma ligands has been performed in several manners: tumor cell lines, isolated organ bath, in vivo animal model. The last is not considered in the present paper because this method studied the physiological role of sigma receptors. The studies carried out in tumor cell lines involved the role of sigma receptors in tumors progression while, although isolated organ bath experiment employed physiological samples, the pharmacokinetic properties of ligands, a strictly requirement for the in vivo assays, did not affect the pharmacodynamic properties of tested compounds. The advances in the above mentioned assays have been reported.

Synthesis and characterization of environment-sensitive fluorescent ligands for human 5-HT1A receptors with 1-arylpiperazine structure.

A series of "long-chain" 1-(2-methoxyphenyl)piperazine derivatives containing an environment-sensitive fluorescent moiety (4-amino-1,8-naphthalimide, 4-dimethylaminophthalimide, dansyl) was synthesized. The compounds displayed very high to moderate 5-HT(1A) receptor affinity and good fluorescence properties. 6-Amino-2-[5-[4-(2-methoxyphenyl)-1-piperazinyl]pentyl]-1H-benz[de]isoquinoline-1,3(2H)-dione (4) combined very high 5-HT(1A) receptor affinity (K(i) = 0.67 nM), high fluorescence emission in CHCl(3), and undetectable fluorescence emission in aqueous solution. It was evaluated for its ability to visualize 5-HT(1A) receptors overexpressed in CHO cells by fluorescence microscopy.

Multi-drug-resistance-reverting agents: 2-aryloxazole and 2-arylthiazole derivatives as potent BCRP or MRP1 inhibitors.

2-Aryloxazole and 2-arylthiazole derivatives were evaluated for their inhibitory activity toward P-glycoprotein (P-gp) as well as their selectivity toward other ABC transporters involved in multi-drug resistance such as BCRP and MRP1. These derivatives have 6,7-dimethoxytetrahydroisoquinoline or cyclohexylpiperazine moieties, which are the same basic nuclei of the potent P-gp inhibitors MC70 (EC(50)=0.05 microM) and PB28 (EC(50)=0.55 microM), respectively. The results demonstrate that 2-aryloxazole and 2-arylthiazole derivatives, planned as cycloisosteres of MC70, were found to be less potent than the reference compound in inhibiting P-gp. These compounds were evaluated for their BCRP and MRP1 inhibitory activities. In particular, 6,7-dimethoxytetrahydroisoquinoline derivatives, unsubstituted, 3-Br, 3-Cl, and 3-OCH(3) 2-aryloxalzole derivatives showed the best BCRP inhibitory activity (EC(50) range: 0.24-0.46 microM). In contrast, all cyclohexylpiperazine derivatives except one (EC(50)=0.56 microM), showed decreased BCRP inhibitory activity. All compounds tested were unable to inhibit the MRP1 pump, with the exception of the 2-OCH(3) and 4-OCH(3) derivatives of the 6,7-dimethoxytetrahydroisoquinoline series, which displayed high MRP1 inhibitory activity (EC(50)=0.84 and 0.90 microM, respectively).

Bicalutamide failure in prostate cancer treatment: involvement of Multi Drug Resistance proteins.

Prolonged bicalutamide treatment induced pathology regression although relapses with a more aggressive form of prostate cancer have been observed. This failure could be due to androgen receptor mutation. In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins (MRPs) that extrude the androgen antagonist from the cell membrane. As experimental models androgen-dependent (LnCap) and androgen-independent (PC-3) prostate cancer cell lines have been employed. Bicalutamide has been tested in the cell lines mentioned above in the absence and in the presence of MC18, our potent P-glycoprotein/BCRP/MRP1 inhibitor. The results displayed that bicalutamide antiproliferative effect at 72 h was ameliorated in LnCap cells (EC(50) from 51.9+/-6.1 microM to 17.8+/-2.6 microM in the absence and in the presence of MC18, respectively) and restored in PC-3 cells (EC(50) from 150+/-2.4 microM to 60+/-3.5 microM in the absence and in the presence of MC18, respectively). Moreover, we established the contribution of each transporter employing stable transfected cells (MDCK) overexpressing P-glycoprotein or BCRP or MRP1 pump. The results displayed that P-glycoprotein and BCRP were involved in bicalutamide efflux while MRP1 was unable to bind the antiandrogen drug.

Effect of some P-glycoprotein modulators on Rhodamine-123 absorption in guinea-pig ileum.

Several reference compounds such as Cyclosporin A, Tamoxifen, Verapamil, and our compound 1, known as P-gp modulators, have been tested for their P-gp modulating activity in isolated organ bath. Compound 1 showed the best result in organ bath experiment (EC(50)=14.7 microM), Cyclosporin A and Tamoxifen displayed EC(50)=25.2 and 39.4 microM, respectively.

PB183, a sigma receptor ligand, as a potential PET probe for the imaging of prostate adenocarcinoma.

PB183, a non-selective sigma receptor ligand displaying high sigma-1 and sigma-2 receptor affinity, was evaluated in prostate tumour cell lines for its suitability as PET radiotracer. The pharmacodynamic and pharmacokinetic properties suggested PB183 as a potential PET radiotracer to visualize prostate adenocarcinoma.

4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators.

Starting from lead compound 1 (EC(50)=1.64 microM), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d displayed the best results (EC(50)=0.05 microM). The corresponding amide 3d was found less active (EC(50)=3.5 microM) ascertaining the importance of basicity in this series whilst the presence of hydroxy or methoxy substituents seems to be negligible. In the naphthyl series, both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible. Moreover, the lipophilicity did not influence the P-gp inhibition activity of each series. Specific biological assays have been carried out to establish the P-gp interacting mechanism of tested compounds discriminating between substrates and inhibitors. Moreover, compound 4d displayed a potent P-gp inhibition activity with good selectivity towards BCRP pump.

Tritium radiolabelling of PB28, a potent sigma-2 receptor ligand: pharmacokinetic and pharmacodynamic characterization.

A potent sigma-2 receptor ligand, known as PB28, was tritium radiolabelled and biologically evaluated. The results showed that [(3)H]PB28 and the corresponding unlabelled PB28 had superimposed pharmacodynamic properties. This radioligand appears as a potential candidate for receptor binding and in living cells assays.

Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives.

The development of small molecules as P-gp modulating agents and SAR studies on these ligands represented the aim of the present work. A series of 6,7-dimethoxytetrahydroisoquinoline derivatives was prepared and their ability to inhibit P-gp activity has been evaluated. The basic nucleus of these compounds, common to the best P-gp inhibitors such as Tariquidar and Elacridar, has been functionalized with no-basic moiety from our studied sigma receptor ligands displaying potent P-gp inhibition. The best results were obtained for compounds 3c and 3a (EC(50)=1.64 and 4.86 microM, respectively) and these results were remarkable because Elacridar showed in the same biological evaluation similar inhibitory activity (EC(50)=2 microM). SAR studies displayed that the removal of double bond on the spacer or its shifting into tetraline ring decreased the P-gp inhibiting activity. Moreover, the P-gp inhibition mechanism of tested compounds was investigated by three selected biological experiments. The results displayed that only compound 3c was P-gp inhibitor as Elacridar, while compound 3a and reference compounds Cyclosporin A and Verapamil modulated P-gp activity saturating the efflux pump as substrates. Flow cytometry studies carried out in Doxorubicin resistant breast cancer cell line (MCF7/Adr) confirmed that compound 3c increased Doxorubicin cell accumulation 5.7-fold. In addition, in MCF7/Adr, antiproliferative effect of 5 microM Doxorubicin shifted from 5% to 95% when co-administered with compound 3c (20 microM). The present study suggested a new class of small molecules displaying P-gp inhibitor activity differing from reference compounds Elacridar and Tariquidar for a simplified, and in the meantime, efficacious no-basic moiety.

Design and evaluation of naphthol- and carbazole-containing fluorescent sigma ligands as potential probes for receptor binding studies.

Some 3,3-dimethyl-1-(3-naphthylpropyl)piperidine and 1-cyclohexyl-4-(3-naphthylpropyl)piperazine derivatives, structurally containing naphthol as a fluorescent moiety, were prepared for being potentially used as fluorescent sigma ligands. Structurally related analogs were also prepared, where the naphthalene nucleus was replaced by the fluorescent carbazole moiety and chain length was varied. For all compounds the in vitro affinities toward sigma receptors and Delta8-Delta7 sterol isomerase site were measured, and the fluorescent properties were determined. Compound 19 gave the best results both for sigma receptor affinities (sigma1, Ki = 6.78 nM and sigma2, Ki = 26.4 nM) and fluorescence features; thus, it was chosen for in vitro saturation binding analysis at sigma receptors. The good results obtained in such assay suggested that the fluorescent compound 19 could be used instead of a radioligand in "green" binding assays.